PROJECT SUMMARY Some head and neck squamous cell carcinoma (HNSCC) patients see pronounced clinical responses with immunotherapeutic intervention. These successes demonstrate the power of therapeutically (re-)activating antitumor T cells to control cancer, but are insufficient, considering the many more patients who do not experience clinical benefit when provided the same treatments. Increasing evidence shows two main barriers to immunotherapy?s success against HNSCC: (i) the tumor?s often poor antigenicity, which limits the generation of antitumor immunity, (ii) suppressive mechanisms that enforce immune tolerance within tumors. This P01?s goal is to address these two barriers: Projects 1 and 2 aim to identify ways that increase HNSCC antigenicity; this Project aims to direct enhanced antigenicity into a successful antitumor immune response. To this end, we will harness myeloid cell populations and their crosstalk with T cells. When considering myeloid cells, we are particularly interested in tumor-infiltrating dendritic cell (DC) populations, since they can present tumor-antigen to T cells and license the execution of T cell-mediated tumor control. We will initially assess three drugs, which we carefully chose based on their use in the clinic, their relevance to the other Projects, our initial data, and their ability to target multiple tumor-associated components. Namely, we will study: (1) 5?azacytidine since it augments tumor cell antigenicity in HNSCC patients and mice (see also Project 1); (2) anti-PD-1 mAb since it can activate T cells to produce key antitumor cytokines such as IFN-gamma and is efficacious in some HNSCC patients; (3) agonistic anti-CD40 mAb since it can induce a DC subset to produce the effector cytokine IL-12, which fosters antitumor T cell activity. Therefore, these drugs may be rationally combined to promote tumor control by targeting tumor cells, adaptive and innate immune cells simultaneously. We will also analyze other drugs during the 5-year project, based in part on findings from Projects 1 and 2. To define drug effects on myeloid?lymphoid cell crosstalk, we will leverage an ongoing clinical trial and animal models that recapitulate key features of the human disease. We will also use two complementary single cell resolution approaches: single cell RNA sequencing (scRNAseq) and single cell imaging (scIMAG). scRNAseq will provide an unbiased view of drug-induced immune changes in human and mouse lesions, and permit comparison across species to facilitate HNSCC translational research. scIMAG will provide information about drug-induced immune responses over time and within the geographical context of the tumor. We hypothesize that these approaches can identify mechanisms of drug-induced myeloid?lymphoid cell crosstalk, which are causally linked to HNSCC control and can be harnessed for therapy. We should be well positioned to perform the proposed work, having experience with studying immune cells and immunotherapy drugs, and having validated the tools that will be exploited in this project. Also, we have assembled a team of experts who will contribute to the project by providing key expertise in HNSCC immunotherapy, cell profiling, imaging, and statistics.